A carbofuran-resistant strain (CAS) showed ca. 41.1- and 15.1-fold resistance compared to a susceptible strain (SUS) and a non-selected field strain (FM), respectively. Enhanced activities of carboxylesterase and P450 were found as ca. 3- and 1.6-fold higher in CAS strain, suggesting these enzymes play a minor role in carbofuran resistance. Interestingly, the insensitivity of acetylcholinesterase (AChE) to carbofuran was revealed to be ca. 5.5- and 3.7-fold higher in CAS strain compared to, indicating that AChE insensitivity mechanism is associated with carbofuran resistance. In the western blot analysis, two kinds of AChEs were found and type-1 AChE (Nlace1) was identified as the major AChE in N. lugens. The open reading frame of Nlace1 is composed of 2,106 bp (ca. 78 Kd) and revealed 52.5% and 24.3% identity compared with Nephotettix cincticeps and Drosophila melanogaster, respectively. In the screening of point mutations, four amino acid substitutions (G119A, F/S330Y, F331I and H332L) were identified in the CAS strain that likely contribute to the AChE insensitivity. The allele frequencies of these mutations increased in the survived populations following the selection by LC50 of carbofuran, confirming that they are in fact associated with reduced sensitivity to carbofuran in N. lugens. These point mutation can be useful for the monitoring of resistance levels in conjunction with QS methods.